RhoU undergoes K48-linked ubiquitination. (A) Anti-PAK4 antibody was used to precipitate phospho-PAK4 (S474) from GFP or GFP-RhoU–transfected MDA-MB-231 cell lysates. (B) An in vitro kinase assay was performed using 1 µg purified GST-PAK4 and 5 µg GST-RhoU. (Left) Coomassie staining demonstrates purified protein input. (Right) Phosphorylation was detected by autoradiography. The asterisk denotes a nonspecific band always observed in PAK4 kinase assays. (C) Lysates were made from control- and PAK4 shRNA-expressing MDA-MB-231 cells and analyzed by immunoblotting with anti-PAK4 and RhoU antibodies. GAPDH was used as a loading control. Autoradiographs were quantified using ImageJ software, and the relative intensity of the PAK4 and RhoU signal was normalized to the loading control. The results shown are the means ± SEM of at least three independent experiments. ***, P < 0.001. (D) mRNA expression of RhoU and β-actin in control and PAK4 knockdown MDA-MB-231 cells as determined by RT-PCR. The relative intensity of the RhoU signal was normalized to the levels of β-actin. The results shown are the means ± SEM of three independent experiments. ns, not significant. (E) Anti-HA antibody coimmunoprecipitation of HA-Ub species and myc-RhoU from HEK-293 cells expressing HA-RhoU and either HA-Ub WT, HA-Ub K48R, or HA-Ub K63R. (F) Anti-Myc antibody coimmunoprecipitation of myc-Ub and HA-RhoU from HEK-293 cells expressing myc-Ub, HA-RhoU point mutants (expression plasmids with a lysine to arginine substitution at positions 177, 248, or both 177 and 248 as indicated), and GFP-PAK4. Ctrl, control.

RhoU interacts with the E3 Ub ligase component Rab40A and is protected from ubiquitination by PAK4. (A) Protein microarray of RhoU–E3 ligase interactions. Column headers are as follows: ID, GenBank accession number; AvgML, duplicate-summarized, loess-normalized, log2-transformed fluorescence intensity values obtained from the experimental array, minus those obtained from the negative control array; SD, standard deviation of the difference. P-value was calculated using a paired two-tailed t test for significance. P < 0.05 was considered to be statistically significant. (B) GST-RhoU pull-down of GFP-Rab40A and T7–Cullin 5 (Cul5). Samples were analyzed by anti-GFP and T7 immunoblotting and Coomassie staining. Representative of three independent experiments. (C) Lysates were made from HEK-293 cells expressing GFP-Rab40A and/or T7–Cullin 5 (Cul5) or T7–Cullin 5 K799R (Cul5 KR) and immunoblotted with anti-GFP and RhoU antibodies. GAPDH was used as a loading control. UT, untransfected. Autoradiographs were quantified using ImageJ software, and the relative intensity of the RhoU signal was normalized to the loading control. The results shown are the means ± SEM of at least three independent experiments. ***, P < 0.001. ns, not significant. (D) Coimmunoprecipitation of myc-Ub and HA-RhoU from HEK-293 cells expressing myc-Ub, HA-RhoU, and either GFP-Rab40A, T7–Cullin 5 (Cul5), or GFP-Rab40A and T7–Cullin 5. (E) Lysates from MDA-MB-231 cells where PAK4 had been silenced or PAK4 had been silenced in combination with Rab40A after 48 h and immunoblotted with anti-PAK4, RhoU, and Rab40A antibodies. GAPDH was used as a loading control. Autoradiographs were quantified using ImageJ software, and the relative intensity of the RhoU signal was normalized to the loading control. The results shown are the means ± SEM of at least three independent experiments. *, P < 0.05; ***, P < 0.001. (F) Anti-Myc antibody coimmunoprecipitation of myc-Ub and HA-RhoU from HEK-293 cells expressing myc-Ub, HA-RhoU domain mutants, and GFP-PAK4. (G) Anti-Myc antibody coimmunoprecipitation of myc-Ub and HA-RhoU from HEK-293 cells expressing myc-Ub, HA-RhoU, and GFP-tagged PAK4 mutants. Ctrl, control.